Gamma-aminobutyric type A receptors (GABAARs) are the principle sites of synaptic inhibition in the brain. It is unclear how neurons regulate the accumulation of synaptic GABAAR subtypes at inhibitory synapses. The impact these processes have on behavior also remains poorly understood. We hypothesize that the synaptic accumulation of GABAARs is dependent on the direct binding of conserved amino acids within the intracellular domains of the 11 and 12 subunits to the inhibitory postsynaptic scaffold protein gephyrin. This interaction is subject to dynamic modulation via the phosphorylation of serine residues within these intracellular domains, a process that determines both receptor number at synaptic sites and the efficacy of synaptic inhibition. Selectively disrupting the binding of GABAARs to gephyrin in the cortex of mice leads to deficits in paired-pulse inhibition, reminiscent of the sensorimotor deficits seen in humans with schizophrenia. To test this central hypothesis we will focus on four specific aims: 1. To test the hypothesis that selective binding of gephyrin to a conserved motif within the receptor 11 and 12 subunits mediates binding to the inhibitory scaffold gephyrin. We will delineate the residues within the 11 and 12 subunits that are responsible for gephyrin binding and then use isothermal titration calorimetry to determine the affinities of these subunits for gephyrin. 2. To test the hypothesis that inhibiting the interaction of gephyrin with the GABAAR 1 subunits modifies both receptor accumulation at synaptic sites and the efficacy of neuronal inhibition. We will compare the properties of miniature inhibitory postsynaptic currents in neurons expressing fluorescent wild-type and mutant 11 subunits, which have reduced capacity to bind gephyrin then go on to examine the effects of adeno-associated viruses that we have created on the synaptic clustering of endogenous GABAARs containing 11/12 subunits. 3. To test the hypothesis that phosphorylation of GABAAR 1 subunits by casein kinase 1 acts as a molecular switch to regulate their interaction with gephyrin. We will assess the role of casein kinase 1-dependent phosphorylation in regulating the membrane trafficking of GABAAR-containing 11/12 subunits, their accumulation at inhibitory synapses and the efficacy of neuronal inhibition. 4. To test the hypothesis that inhibiting the binding of gephyrin to GABAAR 1 subunit in the cortex leads to schizophrenic-like behavior in mice. We will evaluate if deficits in paired pulse inhibition are reversed by antipsychotics and subtype-selective benzodiazepines. We will also assess if blocking GABAAR clustering in the cortex induces other schizophrenia-related phenotypes in the absence and presence of psychostimulants. PUBLIC HEALTH RELEVANCE: The experiments in this proposal are designed to provide mechanistic insights into the cellular processes that control the accumulation and stabilization of GABAARs at synaptic sites and determine how they impact on the pathology of schizophrenia. This study has the potential to make contributions to the development of novel therapeutics to alleviate schizophrenia in addition to a plethora of other neuropsychiatric disorders in which modified inhibition is of significance.